FTL3, as well as KIT, FMS and PDGFR, is a protein of a class III receptor tyrosine kinase (RTK), and is presumed to be involved in the hematopoietic system (Rosnet, O. et al., 1991, Genomics 9:380-385; Rosnet, O. et al., 1991, Oncogene, 6:1641-1650; Matthews, W. et al., 1991, Cell, 65: 1143-1152; Rosnet, O. et al., 1993, Blood, 82:1110-1119). Structurally, RTK has an extracellular region containing five immunoglobulin-like domains, one juxtamembrane region (JM domain), two tyrosine domains (TK1 and TK2) intervened by a kinase insert domain (KI domain), and the C-terminal domain. FLT3 is strongly expressed in the hematopoietic stem cells as well as the brain, placenta and liver (Rosnet, O. et al., 1991, Oncogene 6:1641-1650; Matthews, W. et al., 1991, Cell, 65: 1143-1152; Rosnet, O. et al., 1993, Blood, 82: 1110-1119; Rusten, L. S., 1996, 87: 1317-1325). A ligand for FLT3 (FL) is expressed from stromal cells in the bone marrow, and present in a membrane-bound or soluble form. This ligand stimulates stem cells independently or together with other cytokines (Hannum, C. et al., 1994, Nature, 368: 643-648; McKenna, H. J. et al., 1995, Blood, 86: 3413-3420; Hirayama, F., 1995, Blood, 85: 1762-1768; Lisovsky, M. et al., 1996, Leukemia, 10: 1012-1018). Therefore, the ligand-receptor interaction between FL and FLT3 is thought to play an important role in the hematopoietic system.
On the other hand, high levels of FLT3 expression are observed in most of the specimens from patients with acute myeloid leukemia (AML) or acute chronic lymphocytic leukemia (ALL). High levels of FLT3 expression are also found in the patients with chronic myeloid leukemia (CML). FL is known to stimulate the proliferation of AML cells more prominently than AML cells (Piacibello, W. et al., 1995, Blood, 86: 4105-4114; Stacchini, A. et al., 1996, Leukemia, 10: 1584-1591; Lisovsky, M. et al., 1996, Blood, 88: 3987-3997; Birg, F. et al., 1992, Blood, 80: 2584-2593, Dehmel, U. et al., 1996, Leukemia, 10: 261-270). This indicates that FLT3 has a function specific to myeloid cells. In several leukemia-lymphoma cell lines, FLT3 and FL are co-expressed (DaSilva, N. et al., 1994, Leukemia, 8: 885-888; Meierhoff, G. 1995, Leukemia, 9: 1368-1372), suggesting their autocrine or paracrine mechanism.
Mutations in cytokine receptors that occur in the process of tumorigenesis have received attention in recent years. To date, mutations in c-fms and c-kit in human leukemia have been reported (Lowenverg, B. and Touw, I. P., 1993, Blood, 81: 281-292). Murine NIH3T3 cells transfected with a mutant c-fms undergo ligand-independent transformation (Roussel, M. et al., 1988, Cell, 55: 979-988). M-CSF, a ligand for fms, increases cell proliferation only slightly in the most of leukemic patients. Thus, the significance of FMS mutation is still unknown (Lowenberg, B. and Touw, I. P., 1993, Blood, 81: 281-292). KIT and its ligand SCF stimulate proliferation of leukemia and stem cells (Lowenberg, B. and Touw, I. P., 1993, Blood, 81: 281-292; Witte, O., 1990, Cell, 63: 5-6). However, mutations in c-kit gene have been found in the mast cell leukemic cell lines, which mutations have not been fully identified in clinical samples (Tsujimura, T. et al., 1994, Blood, 83: 2619-2626; Kitayama, H., 1996, Blood, 88: 995-1004; Tsujimura, Y. et al., 1996, Blood, 87: 273-283).
Recently, somatic mutations in FLT3 were found in AML patients (Nakao, M. et al., 1996, Leukemia, 10: 1911-1918). In these mutants, internal tandem duplication (ITD) was found in the region coding for the JM domain of the FLT3 gene. The duplicated sequences predominantly contain exon 11/12 and intron 11, though varying in length in each sample, and they commonly have an extended JM domain which is translatable in a protein due to an extended in-frame open reading frame.
FLT3 mutations are found in about 20% of AML patients and about 3% of patients with myelodysplasia syndrome (MDS), but not in patients with chronic myeloid leukemia (CML) or lymphocytic blood cancer (Yokota, S. et al., 1997, Leukemia 11: 1605-1609). To the inventors' knowledge, mutant FLT3 genes containing ITD (hereinafter referred to as “FLT3/ITD”) are found in some AML patients when the tumor recurs, even though they are not found by the early diagnosis, suggesting that FLT3/ITD is responsible for progression of leukemia. However, the role of FLT3/ITD in the progression of leukemia has not been reported so far.